Determination of tetrodotoxin in puffer-fish by liquid chromatography-electrospray ionization mass spectrometry

A simple and reliable method using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) for the determination of tetrodotoxin in the puffer-fish has been developed. The LC separation was performed on a Shodex RSpak NN-414 column (15 cm x 4.6 mm id) using 20 mM ammonium acetate-methanol (75 + 25) as the mobile phase at a flow rate of 0.5 ml min(-1). The positive ionization produced the typical [M + H]+ molecular ion of tetrodotoxin (m/z 320). The calibration graph for tetrodotoxin was rectilinear from 0.01 to 1 microg ml(-1) with selected ion monitoring (SIM). Tetrodotoxin was extracted with 0.1% acetic acid by heating in a boiling water-bath and the extracts were cleaned up on a Bond Elut C18 (500 mg) cartridge. The recoveries of the tetrodotoxin from the puffer-fish fortified at 1 microg g(-1) were 77.7-80.7% and the detection limit was 0.1 microg g(-1) (equivalent to ca. 0.5 mouse units per gram).